The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.     

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.     

Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-D gel electrophoresis and MALDI-TOF MS

Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.     

Oncogenic role of mortalin contributes to ovarian tumorigenesis by activating the MAPK–ERK pathway

Mortalin is frequently overexpressed in human malignancies. Previous studies have suggested that mortalin contributes to ovarian cancer development and progression, but further investigation is warranted. The aim of this study is to elucidate the mechanism of mortalin in ovarian cancer development and progression. In this study, lentivirus‐delivered mortalin short hairpin RNA (shRNA) was used to knockdown mortalin expression in A2780 and A2780/cis ovarian cancer cell lines, and lentiviral mortalin‐pLVX‐AcGFP was used to generate mortalin‐overexpressing cell lines. The results demonstrated that decreased mortalin expression reduced ovarian cancer cell proliferation, colony formation, migration and invasion by Cell Counting Kit‐8 assay, colony formation assay, wounding healing assay and Transwell cell invasion assay, respectively. Flow cytometry results suggested that mortalin promotes the G1 transition, leading to faster restoration of a normal cell‐cycle distribution. Cell‐cycle proteins, including C‐myc and Cyclin‐D1, significantly increased, and Cyclin‐B1 remarkably decreased upon mortalin down‐regulation. Western blot analysis showed that mortalin knockdown significantly decreased p‐c‐Raf and phospho‐extracellular–regulated protein kinases (p‐ERK1/2) pathways but not the Jun N‐terminal kinase pathway, whereas mortalin overexpression had the opposite effect. Taken together, these results indicate that mortalin is an oncogenic factor, and mitogen‐activated protein kinase‐ERK signalling pathway activation by mortalin may contribute to ovarian cancer development and progression.     

miR-421 promotes apoptosis and suppresses metastasis of osteosarcoma cells via targeting LTBP2

Increasing evidence has confirmed that microRNAs (miRs) are involved in tumor development and progression. A previous study reported that miR-421 could serve as a diagnostic marker in patients with osteosarcoma (OS). The present study explored the potential roles of miR-421 in the regulation of cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition of OS cells. Our results showed that miR-421 was upregulated in OS tissues and cell lines (MG63, U2OS, HOS, and Saos-2) compared with the corresponding adjacent tissues or human osteoblast cells hFOB1.19, while the latent transforming growth factor β-binding protein 2 (LTBP2) expression was reduced. In MG63 and U2OS cells, CCK8 assay displayed that cell proliferation was repressed by the miR-421 inhibitor, conversely increased by miR-421 mimics. Inhibition of miR-421 promoted cell apoptosis rate, caspase 3 activity, cleaved-caspase 3 (c-caspase 3) expression, and Bax/Bcl-2 ratio, restoration of miR-421 showed the opposite functions. Suppression of miR-421 blocked migration and invasion, whereas miR-421 overexpression promoted the migration and invasion of MG63 and U2OS cells. In addition, real-time polymerase chain reaction and Western blot analysis revealed that miR-421 negatively regulated E-cadherin expression, and positively regulated the expression of N-cadherin and vimentin. The luciferase reporter assay determined that miR-421 could target LTBP2-3′-UTR, and LTBP2 expression was regulated negatively by miR-421 both in mRNA and protein levels. Depletion of LTBP2 partly abolished the biological functions of miR-421 inhibitor in OS. In conclusion, miR-421 plays an oncogenic role in OS via targeting LTBP2, suggesting that miR-421 may be a potential therapeutic target against OS.     

Secretion modification region-derived peptide disrupts HIV-1 Nef's interaction with mortalin and blocks virus and Nef exosome release

Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4⁺ T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef''s SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties.     

Mortalin/GRP75 Binds to Complement C9 and Plays a Role in Resistance to Complement-dependent Cytotoxicity

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.     

Apoptotic cell death in TrkA-overexpressing cells: kinetic regulation of ERK phosphorylation and caspase-7 activation

The TrkA tyrosine kinase is activated by autophosphorylation in response to NGF, and plays an important role in cell survival, differentiation, and apoptosis. To investigate its role in cell fate determination, we produced stable TrkA-inducible SK-N-MC and U2OS cell lines using the Tet-On system. Interestingly, TrkA overexpression induced substantial cell death even in the absence of NGF, by stimulating ERK phosphorylation and caspase-7 activation leading to PARP cleavage. TrkA-mediated cell death was shown by the annexin-V binding assay to be, at least in part, apoptotic in both SK-N-MC and U2OS cells. Furthermore, the truncated form (p18) of Bax accumulated in the TrkA-induced cells, suggesting that TrkA induces mitochondria-mediated apoptosis. NGF treatment augmented the cell death induced by TrkA overexpression. This TrkA-induced cell death was blocked by the tyrosine kinase inhibitors, K-252a and GW441756. Moreover, TrkA overexpression inhibited long-term proliferation of both the neuronal SK-N-MC cells and the non-neuronal U2OS cells, suggesting a potential role of TrkA as a tumor suppressor.     

Calbindin 2 (CALB2) regulates 5-fluorouracil sensitivity in colorectal cancer by modulating the intrinsic apoptotic pathway

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Δψ(m)) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.     

MiRNA-26b regulates the expression of cyclooxygenase-2 in desferrioxamine-treated CNE cells

Here we report that miR-26b is involved in COX-2 overexpression in desferrioxamine (DFOM)-treated carcinoma of nasopharyngeal epithelial (CNE) cells. The level of miR-26b in DFOM-treated CNE cells is inversely proportional to the expression level of the COX-2 protein. Overexpression of miR-26b in DFOM-treated CNE cells inhibits cell proliferation. A luciferase reporter gene experiment suggests that the 3′ untranslated region of COX-2 carries a binding site for miR-26b. Overexpression of miR-26b marginally reduces the levels of COX-2 protein in DFOM-treated CNE cells. Moreover, knockdown of COX-2 expression had a similar effect to overexpression of miR-26b. Taken together, these results suggest that miR-26b regulates COX-2 expression in DFOM-treated cells.     

The calcium binding protein ALG-2 binds and stabilizes Scotin, a p53-inducible gene product localized at the endoplasmic reticulum membrane

ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG101, respectively. An in vitro synthesized C-terminal fragment of Scotin bound specifically to immobilized recombinant ALG-2 and tagged ALG-2 and Scotin were shown by immunoprecipitation to interact in MCF7 and U2OS cell lines. Furthermore ALG-2 bound to endogenous Scotin in extracts from mouse NIH3T3 cells. Overexpression of ALG-2 led to accumulation of Scotin in MCF7 and H1299 cells. In vitro and in vivo binding of ALG-2 to Scotin was demonstrated to be strictly calcium dependent indicating a role of this interaction in calcium signaling pathways.     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

Polo-like kinase-2 is required for centriole duplication in mammalian cells

Centriole duplication initiates at the G1-to-S transition in mammalian cells and is completed during the S and G2 phases. The localization of a number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling the centriole duplication cycle. Here we show that the human Polo-like kinase 2 (Plk2) is activated near the G1-to-S transition of the cell cycle. Endogenous and overexpressed HA-Plk2 localize with centrosomes, and this interaction is independent of Plk2 kinase activity. In contrast, the kinase activity of Plk2 is required for centriole duplication. Overexpression of a kinase-deficient mutant under S-phase arrest blocks centriole duplication. Downregulation of endogenous Plk2 with small hairpin RNAs interferes with the ability to reduplicate centrioles. Furthermore, centrioles failed to duplicate during the cell cycle of human fibroblasts and U2OS cells after overexpression of a Plk2 dominant-negative mutant. These results show that Plk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1-to-S phase transition.     

Expression of the double-stranded RNA-dependent kinase PKR influences osteosarcoma attachment independent growth,migration, and invasion

Osteosarcoma (OS) is a rare, insidious tumor of mesenchymal origin that most often affects children, adolescents, and young adults. While the primary tumor can be controlled with chemotherapy and surgery, it is the lung metastases that are eventually fatal. Multiple studies into the initial drivers of OS development have been undertaken, but few of these have examined innate immune/inflammatory signaling. A central figure in inflammatory signaling is the innate immune/stress-activated kinase double-stranded RNA-dependent protein kinase (PKR). To characterize the role of PKR in OS, U2OS, and SaOS-2 osteosarcoma cell lines were stably transfected with wild-type or dominant-negative (DN) PKR. Overexpression of PKR enhanced colony formation in soft agar (U2OS and SaOS-2), enhanced cellular migration (U2OS), and invasive migration (SaOS-2). In contrast, overexpression of DN-PKR inhibited attachment-independent growth, migration and/or invasion. These data demonstrate a role for inflammatory signaling in OS formation and migration/invasion and suggest the status of PKR expression/activation may have prognostic value.     

BRCA1-induced apoptosis involves inactivation of ERK1/2 activities

Mutation in the BRCA1 gene is associated with an increased risk of breast and ovarian cancer. Recent studies have shown that the BRCA1 gene product may be important in mediating responses to DNA damage and genomic instability. Previous studies have indicated that overexpression of BRCA1 can induce apoptosis or cell cycle arrest at the G(2)/M border in various cell types. Although the activation of JNK kinase has been implicated in BRCA1-induced apoptosis, the role of other members of the mitogen-activated protein kinase family in mediating the cellular response to BRCA1 has not yet been examined. In this study, we monitored the activities of three members of the MAPK family (ERK1/2, JNK, p38) in MCF-7 breast cancer cells and U2OS osteosarcoma cells after their exposure to a recombinant adenovirus expressing wild type BRCA1 (Ad.BRCA1). Overexpression of BRCA1 in MCF-7 cells resulted in arrest at the G(2)/M border; however, BRCA1 expression in U2OS cells induced apoptosis. Although BRCA1 induced JNK activation in both cell lines, there were marked differences in ERK1/2 activation in response to BRCA1 expression in these two cell lines. BRCA1-induced apoptosis in U2OS cells was associated with no activation of ERK1/2. In contrast, BRCA1 expression in MCF-7 cells resulted in the activation of both ERK1/2 and JNK. To directly assess the role of ERK1/2 in determining the cellular response to BRCA1, we used dominant negative mutants of MEK1 as well as MEK1/2 inhibitor PD98059. Our results indicate that inhibition of ERK1/2 activation resulted in increased apoptosis after BRCA1 expression in MCF-7 cells. Furthermore, BRCA1-induced apoptosis involved activation of JNK, induction of Fas-L/Fas interaction, and activation of caspases 8 and 9. The studies presented in this report indicate that the response to BRCA1 expression is determined by the regulation of both the JNK and ERK1/2 signaling pathways in cells.